In the last fifteen years, the important roles of small non-coding RNAs in regulation in all organisms have been recognized. Our laboratory, in collaboration with others, undertook two global searches for non-coding RNAs in E. coli, contributing significantly to the more than 80 regulatory RNAs that are now identified. A large number of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number of these sRNAs in detail. Each sRNA is regulated by different stress conditions, suggesting that the sRNA plays an important role in adapting to stress. We have also examined the mechanism by which Hfq operates to allow sRNAs to act. The lab continues to investigate the in vivo roles of small RNAs, identifying the regulatory networks they participate in and their roles in those networks. Many targets of sRNAs are regulated by more than one sRNA, and our earlier approaches have not allowed us to identify all of the sRNAs regulating a given target. In addition, regulatory proteins expressed at low levels or under special conditions may not be identified as targets for a given sRNA if their transcripts are not reasonably abundant. We have developed and made use of an approach to dealing with these limitations. Translational fusions can easily be created to possible target genes, identified based on the ability of their mRNA to bind Hfq, among other criteria, and the fusions can be rapidly screened with a library of plasmids, each expressing a different sRNA. Bacterial behavior can similarly be screened. We have applied this approach to the study of bacterial motility and biofilm formation, and have also examined the sRNAs regulating a set of important transcriptional regulators and proteins involved in stress responses. We find that multiple sRNAs regulate bacterial motility, many of them by regulating a critical transcriptional activator of flagellar synthesis, flhDC. Two sRNAs positively regulate motility, while at least four down-regulate motility. These provide unexpected new inputs to the well-studied regulation of flagellar synthesis. Bacteria such as E. coli are motile under some circumstances, but in some growth conditions form non-motile biofilms. Not surprisingly, we find that sRNAs play important roles in biofilm formation as well. Our results suggest that both flhDC, the central regulator of motility, and rpoS, encoding the stress sigma factor, act as nodes for regulation by multiple sRNAs. In our screen of transcriptional regulators for sRNA regulation, we find that only a subset of regulators, including Lrp and SoxS, are subject to sRNA regulation, and we are investigating the physiological significance of this extra level of regulation. In another study, a small RNA was found to negatively regulate tolC, the core of multiple drug efflux pumps in E. coli. mutS, a component of the mismatch repair system, is regulated by a small RNA, ArcZ and directly by Hfq; deletion of either affects the level of mutagenesis. The action of these small RNAs depends on the RNA chaperone Hfq, a protein with homology to the Lsm and Sm families of eukaryotic proteins involved in RNA splicing and other functions. Hfq binds both to sRNAs and to mRNAs, and stimulates pairing, but exactly how it does this is not entirely clear. We have now undertaken a careful in vivo dissection of Hfq, in collaboration with G. Storz (NICHD). Interesting hfq alleles were analyzed in vivo with multiple sRNA:mRNA reporters. We have found that the Hfq-dependent sRNAs fall into two classes, defined by their behavior in different Hfq mutants. All of these sRNAs depend on the known sRNA binding site on the proximal face of Hfq for in vivo stability. The larger group, called Class I, is rapidly degraded when used, most likely dependent upon pairing. Mutations in the distal face of Hfq, which disrupt target mRNA binding, stabilize the Class I sRNAs, and mutations in the rim of Hfq, implicated in pairing, destabilize the sRNAs. Class II sRNAs are generally more stable than Class I sRNAs, are not destabilized by the rim mutants, but are by the distal site mutants. These results suggest at least two distinct modes of sRNA binding to Hfq; these different modes of sRNA binding also dictate different modes of binding of the mRNAs for Class I and Class II sRNAs, and suggest that Class II sRNAs are likely to exclude Class I targets from binding to Hfq. These results help to explain previously observed competition between sRNAs and differential effects of different hfq alleles on different sRNA:mRNA pairs. We suggest that the roles of Class I and Class II sRNAs in the cell are somewhat different, reflecting their different stabilities, and are testing this by creating mutants and chimeric sRNAs that alter the behavior of the sRNAs. An unexpected additional direction for our study of translational regulation came from studies of the effect of mutants in pyruvate dehydrogenase (aceE on expression of RpoS, discussed as part of our proteolysis project. In addition to stabilizing RpoS, the aceE mutants greatly increase synthesis of RpoS, independent of the promoter and known sRNA regulators. This increased in translational activation of RpoS may suggest a previously unknown mode of selective translation under metabolic stress. Overall, we have developed highly efficient in vivo tools for studying sRNAs and the networks they reside in. Our focus is increasingly on the role of the sRNAs in complex bacterial behavior, investigations into the mechanism of sRNA function, and dissecting of novel mechanisms for regulating translation initiation.